Computational modeling of inverting glycosyltransferase reaction mechanisms

Abstract

[eng] Carbohydrates, often referred to as sugars, are essential biomolecules found in all living organisms. While they are well-known for their role in providing energy, carbohydrates also play critical roles in various other biological processes, including the formation of structural components like plant cell walls and the facilitation of cell communication. This wide array of functions is related to the remarkable diversity of carbohydrates: they are made up of different types of monomers that can be linked together in numerous configurations, creating molecules of varying complexity and size.

The high diversity of carbohydrates in Nature also requires a multitude of enzymes responsible for catalyzing reactions such as their synthesis, modification, or hydrolysis. These enzymes, known as carbohydrate-active enzymes (CAZymes), are essential for the correct functioning of cells. In this work, we have focused on a specific type of CAZymes: glycosyltransferases (GTs), which accelerate the formation of new glycosidic bonds. In other words, GTs catalyze the creation of linkages between sugars and other carbohydrates, or other biomolecules such as lipids or proteins. More specifically, we have investigated inverting glycosyltransferases, which catalyze the formation of these new bonds with inversion of the anomeric carbon configuration. The “textbook” mechanism followed by inverting GTs is an SN2 one-step reaction in which the acceptor molecule is deprotonated by a general base residue within the active site. However, the specific details of this mechanism can vary across different enzymes.

Our goal is to elucidate the mechanism of selected inverting GTs using computational chemistry methods, primarily classical molecular dynamics, quantum mechanics/molecular mechanics (QM/MM), and metadynamics. Our simulations, in conjunction with experimental results obtained by collaborators from other groups, have revealed the reaction mechanism details of four inverting GTs. These four GTs of interest are of high biomedical and biotechnological importance and are related to the synthesis of protein glycoconjugates. Moreover, some of these enzymes exhibit unique features that set them apart from other inverting GTs, making their study even more compelling. A deeper understanding of their catalytic mechanisms could aid the future development of inhibitors and guide the design of enzyme modifications for biotechnological applications.

The first enzyme we study is α-Mannoside β-1,6-N-acetylglucosaminyltransferase V (or MGAT5), an inverting GT that catalyzes the transfer of GlcNAc to developing N-glycans on the surface of proteins. We reconstructed its Michaelis complex and uncovered the details of its mechanism. The second enzyme is protein O-fucosyltransferase 1 (POFUT1). POFUT1 transfers fucose to threonine or serine residues on epidermal growth

factor-like (EGF-LD) peptides. We determined the mechanism of the enzyme, particularly the deprotonation of the acceptor threonine in absence of a general base residue in the active site, that we found proceeded through an active site asparagine undergoing tautomerization. The third enzyme studied was non-LEE encoded effector protein B 1 (NleB1). NleB1 catalyzes the transfer of GlcNAc to arginine residues on protein death domains, in contrast to the more common N-glycosylation of asparagine. One of the main questions for this enzyme is how an arginine can perform this reaction, as it is poor nucleophile due to the positive charge of its guanidinium ion. We determined its mechanism using path-metadynamics, a modification on the protocol followed in the other sections of the Thesis. Finally, we studied a bacterial N- glycosyltransferase (NGT). Bacterial NGTs glycosylate asparagine on the surface of peptides using UDP-Glc as donor, as opposed to the more ubiquitous OST enzyme. Here, we reconstruct its Michaelis complex and uncover its catalytic mechanism, that operates without a general base.

[cat] Els carbohidrats, també coneguts com a sucres o glúcids, són biomolècules essencials presents a tots els organismes. És ben conegut el seu rol com a font d’energia, però també tenen un rol crucial en altres processos biològics, com la formació de components estructurals com ara parets cel·lulars vegetals, o la comunicació intercel·lular. Aquesta gran varietat de funcions està relacionada amb la seva remarcable diversitat: els carbohidrats estan formats per diferents tipus de monòmers que poden unir-se en nombroses configuracions, creant molècules de d’un ample rang de complexitat.

Aquesta gran diversitat dels carbohidrats a la Natura també requereix una multitud d’enzims responsables de catalitzar reaccions com la seva síntesi, modificació o hidròlisi. Aquests enzims, coneguts com carbohydrate-active enzymes (CAZymes) (enzims actius en carbohidrats), són essencials pel correcte funcionament de les cèl·lules. En aquesta tesi, ens hem centrat en un tipus específic de CAZyme: les glicosiltransferases (GTs), que acceleren la formació de nous enllaços glicosídics. En altres paraules, les GTs catalitzen la creació de noves unions entre sucres i altres carbohidrats, o altres biomolècules com lípids o proteïnes. Més específicament, hem estudiat glicosiltransferases d’inversió, que catalitzen la formació dels nous enllaços amb la inversió de la configuració del carboni anomèric. El mecanisme canònic que normalment segueixen les GTs d’inversió és una reacció d’un pas de tipus SN2, en la que la molècula acceptora és desprotonada per una base general al centre actiu. Malgrat això, els detalls específics de cada mecanisme poden variar segons l’enzim.

El nostre objectiu és descobrir el mecanisme de glicosiltransferases d’inversió seleccionades utilitzant tècniques de química computacional, principalment dinàmica molecular clàssica, mecànica quàntica/mecànica molecular (QM/MM), i metadynamics. Les nostres simulacions, en conjunció amb els resultats experimentals obtinguts per col·laboradors experimentals d’altres grups, han revelat els detalls dels mecanismes de reacció de quatre GTs d’inversió relacionades amb la glicosilació de proteïnes: α- Mannoside β-1,6-N-acetylglucosaminyltransferase V (o MGAT5), protein O- fucosyltransferase 1 (POFUT1), non-LEE encoded effector protein B 1 (NleB1) i una N- glicosiltransferasa bacteriana (NGT).